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Abstract Background The PI3K/Akt pathway is involved in cellular survival pathways by inhibiting apoptotic processes and stimulating cell growth and proliferation. Its negative prognostic value has been proven in many types of cancer. In soft tissue sarcomas, the expression profiles of the PI3K/Akt pathway components are poorly defined and their significance uncertain. We aimed to investigate the prognostic impact of Akt (Akt1) phosphorylated at threonine308 and serine473, Akt2, Akt3, PI3K and PTEN, alone and in coexpression with ER and PgR in non-gastrointestinal stromal tumor soft tissue sarcomas (non-GIST STSs). Patients and methods Tumor samples and clinical data from 249 patients with non-GIST STS were obtained, and tissue microarrays (TMAs) were constructed. Immunohistochemistry (IHC) was used to evaluate marker expression in tumor cells. Results In univariate analyses, the expression levels of p-Akt Thr308 (P = 0.002), Akt2 (P = 0.008) and PI3K (P < 0.001) were significant prognostic factors. In the multivariate analysis, high PI3K expression was an independent negative prognosticator (HR = 1.5, 95% CI = 1.0-2.2, P = 0.042) in addition to advanced age, tumor depth, high malignancy grade, metastasis at diagnosis, surgery and positive resection margins. p-Akt Thr308 expression had strong unfavorable effect in men only (P = 0.009). In contrast, p-Akt Ser473 expression had strong unfavorable impact in women (P = 0.023). PgR-/p-Akt Ser473+ phenotype tended to have less favorable impact in women (P = 0.087), but was the most favorable one in men (P = 0.010). Conclusion Expression of PI3K was significantly associated with aggressive behavior and shorter DSS in non-GIST STSs. The site of Akt phosphorylation seems to have gender-dependent impact on survival in STS patients.

Abstract Background Non-gastrointestinal stromal tumor soft-tissue sarcomas (non-GIST STSs) constitute a heterogeneous group of tumors with poor prognosis. Fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptor-1 (FGFR-1), in close interplay with platelet-derived growth factor-B (PDGF-B) and vascular endothelial growth factor receptor-3 (VEGFR-3), are strongly involved in angiogenesis. This study investigates the prognostic impact of FGF2 and FGFR-1 and explores the impact of their co-expression with PDGF-B and VEGFR-3 in widely resected tumors from non-GIST STS patients. Methods Tumor samples from 108 non-GIST STS patients were obtained and tissue microarrays were constructed for each specimen. Immunohistochemistry was used to evaluate the expressions of FGF-2, FGFR-1, PDGF-B and VEGFR-3. Results In the multivariate analysis, high expression of FGF2 (P = 0.024, HR = 2.2, 95% CI 1.1-4.4) and the co-expressions of FGF2 & PDGF-B (overall; P = 0.007, intermediate; P = 0.013, HR = 3.6, 95% CI = 1.3-9.7, high; P = 0.002, HR = 6.0, 95% CI = 2.0-18.1) and FGF2 & VEGFR-3 (overall; P = 0.050, intermediate; P = 0.058, HR = 2.0, 95% CI = 0.98-4.1, high; P = 0.028, HR = 2.6, 95% CI = 1.1-6.0) were significant independent prognostic indicators of poor disease-specific survival. Conclusion FGF2, alone or in co-expression with PDGF-B and VEGFR-3, is a significant independent negative prognosticator in widely resected non-GIST STS patients.

Abstract Background In recent years, microRNAs (miRNAs) have been found to play an essential role in tumor development. In lung tumorigenesis, targets and pathways of miRNAs are being revealed, and further translational research in this field is warranted. MiR-155 is one of the miRNAs most consistently involved in various neoplastic diseases. We aimed to investigate the prognostic impact of the multifunctional miR-155 in non-small cell lung cancer (NSCLC) patients. Methods Tumor tissue samples from 335 resected stage I to IIIA NSCLC patients were obtained and tissue microarrays (TMAs) were constructed with four cores from each tumor specimen. In situ hybridization (ISH) was used to evaluate the expression of miR-155. Results There were 191 squamous cell carcinomas (SCCs), 95 adenocarcinomas (ACs), 31 large cell carcinomas and 18 bronchioalveolar carcinomas. MiR-155 expression did not have a significant prognostic impact in the total cohort (P = 0.43). In ACs, high miR-155 expression tended to a significant negative prognostic effect on survival in univariate analysis (P = 0.086) and was an independent prognostic factor in multivariate analysis (HR 1.87, CI 95% 1.01 - 3.48, P = 0.047). In SCC patients with lymph node metastasis, however, miR-155 had a positive prognostic impact on survival in univariate (P = 0.034) as well as in multivariate (HR 0.45, CI 95% 0.21-0.96, P = 0.039) analysis. Conclusions The prognostic impact of miR-155 depends on histological subtype and nodal status in NSCLC.

Objective: This study aims to assess the ability of state-of-the-art machine learning algorithms to detect valvular heart disease (VHD) from digital heart sound recordings in a general population that includes asymptomatic cases and intermediate stages of disease progression., Methods: We trained a recurrent neural network to predict murmurs from heart sound audio using annotated recordings collected with digital stethoscopes from four auscultation positions in 2,124 participants from the Tromsø7 study. The predicted murmurs were used to predict VHD as determined by echocardiography., Results: The presence of aortic stenosis (AS) was detected with a sensitivity of 90.9%, a specificity of 94.5%, and an area under the curve (AUC) of 0.979 (CI: 0.963-0.995). At least moderate AS was detected with an AUC of 0.993 (CI: 0.989-0.997). Moderate or greater aortic and mitral regurgitation (AR and MR) were predicted with AUC values of 0.634 (CI: 0.565-703) and 0.549 (CI: 0.506-0.593), respectively, which increased to 0.766 and 0.677 when clinical variables were added as predictors. The AUC for predicting symptomatic cases was higher for AR and MR, 0.756 and 0.711, respectively. Screening jointly for symptomatic regurgitation or presence of stenosis resulted in an AUC of 0.86, with 97.7% of AS cases ( n  = 44) and all 12 MS cases detected., Conclusions: The algorithm demonstrated excellent performance in detecting AS in a general cohort, surpassing observations from similar studies on selected cohorts. The detection of AR and MR based on HS audio was poor, but accuracy was considerably higher for symptomatic cases, and the inclusion of clinical variables improved the performance of the model significantly., Competing Interests: Medsensio AS is a company that provides products for automatic detection of abnormal lung sounds. JR and MJ are employed by Medsensio. HM was working for Medsensio during this research project, and PW was employed for some time by Medsensio after the research project was mostly completed. JR, MJ, and LB owns shares in Medsensio. PW, HM, HS, MJ, TD, JR, SA, AD, JS, and LB have filed two patents for the algorithms outlined in this paper., (© 2024 Waaler, Malbye, Schirmer, Johnsen, Donnem, Ravn, Andersen, Davidsen, Aviles Solis, Stylidis and Bongo.)

Introduction: Non-small cell lung carcinomas (NSCLC) exhibit different microvessel patterns (MVPs). Basal (BA), diffuse (DA) and papillary (PA) patterns show signs of angiogenesis (new blood vessels), while an alveolar pattern indicates that tumors are co-opting existing normal vessels (non-angiogenic alveolar, NAA). NAA tumor growth is known to exist in NSCLC, but little is known about its prognostic impact in different histological subgroups, and about associations between MVPs and immune cell infiltration., Methods: Detailed patterns of angiogenic and non-angiogenic tumor growth were evaluated by CD34 immunohistochemistry in whole tissue slides from 553 surgically treated patients with NSCLC stage I-IIIB disease. Associations with clinicopathological variables and markers related to tumor immunology-, angiogenesis- and hypoxia/metabolism were explored, and disease-specific survival (DSS) was analyzed according to histological subtypes., Results: The predominant MVP was angiogenic in 82% of tumors: BA 40%, DA 34%, PA 8%, while a NAA pattern dominated in 18%. A contribution of the NAA pattern >5% (NAA+), i.e., either dominant or minority, was observed in 40.1% of tumors and was associated with poor disease-specific survival (DSS) ( p =0.015). When stratified by histology, a significantly decreased DSS for NAA+ was found for adenocarcinomas (LUAD) only ( p < 0.003). In multivariate analyses, LUAD NAA+ pattern was a significant independent prognostic factor; HR 2.37 (CI 95%, 1.50-3.73, p < 0.001). The immune cell density (CD3, CD4, CD8, CD45RO, CD204, PD1) added prognostic value in squamous cell carcinoma (LUSC) and LUAD with 0-5% NAA (NAA-), but not in LUAD NAA+. In correlation analyses, there were several significant associations between markers related to tumor metabolism (MCT1, MCT4, GLUT1) and different MVPs., Conclusion: The NAA+ pattern is an independent poor prognostic factor in LUAD. In NAA+ tumors, several immunological markers add prognostic impact in LUSC but not in LUAD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Paulsen, Andersen, Rakaee, Pedersen, Lombardi, Pøhl, Kilvaer, Busund, Pezzella and Donnem.)

Importance: Currently, predictive biomarkers for response to immune checkpoint inhibitor (ICI) therapy in lung cancer are limited. Identifying such biomarkers would be useful to refine patient selection and guide precision therapy., Objective: To develop a machine-learning (ML)-based tumor-infiltrating lymphocytes (TILs) scoring approach, and to evaluate TIL association with clinical outcomes in patients with advanced non-small cell lung cancer (NSCLC)., Design, Setting, and Participants: This multicenter retrospective discovery-validation cohort study included 685 ICI-treated patients with NSCLC with median follow-up of 38.1 and 43.3 months for the discovery (n = 446) and validation (n = 239) cohorts, respectively. Patients were treated between February 2014 and September 2021. We developed an ML automated method to count tumor, stroma, and TIL cells in whole-slide hematoxylin-eosin-stained images of NSCLC tumors. Tumor mutational burden (TMB) and programmed death ligand-1 (PD-L1) expression were assessed separately, and clinical response to ICI therapy was determined by medical record review. Data analysis was performed from June 2021 to April 2022., Exposures: All patients received anti-PD-(L)1 monotherapy., Main Outcomes and Measures: Objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) were determined by blinded medical record review. The area under curve (AUC) of TIL levels, TMB, and PD-L1 in predicting ICI response were calculated using ORR., Results: Overall, there were 248 (56%) women in the discovery cohort and 97 (41%) in the validation cohort. In a multivariable analysis, high TIL level (≥250 cells/mm2) was independently associated with ICI response in both the discovery (PFS: HR, 0.71; P = .006; OS: HR, 0.74; P = .03) and validation (PFS: HR = 0.80; P = .01; OS: HR = 0.75; P = .001) cohorts. Survival benefit was seen in both first- and subsequent-line ICI treatments in patients with NSCLC. In the discovery cohort, the combined models of TILs/PD-L1 or TMB/PD-L1 had additional specificity in differentiating ICI responders compared with PD-L1 alone. In the PD-L1 negative (<1%) subgroup, TIL levels had superior classification accuracy for ICI response (AUC = 0.77) compared with TMB (AUC = 0.65)., Conclusions and Relevance: In these cohorts, TIL levels were robustly and independently associated with response to ICI treatment. Patient TIL assessment is relatively easily incorporated into the workflow of pathology laboratories at minimal additional cost, and may enhance precision therapy.

Background: due to emerging therapeutics targeting KRAS G12C and previous reports with conflicting results regarding the prognostic impact of KRAS and KRAS G12C in non-small cell lung cancer (NSCLC), we aimed to investigate the frequency of KRAS mutations and their associations with clinical characteristics and outcome. Since mutation subtypes have different preferences for downstream pathways, we also aimed to investigate whether there were differences in outcome according to mutation preference for the Raf, PI3K/Akt, or RalGDS/Ral pathways., Methods: retrospectively, clinicopathological data from 1233 stage I-IV non-squamous NSCLC patients with known KRAS status were reviewed. KRAS ' associations with clinical characteristics were analysed. Progression free survival (PFS) and overall survival (OS) were assessed for the following groups: KRAS wild type (wt) versus mutated, KRAS wt versus KRAS G12C versus KRAS non-G12C, among KRAS mutation subtypes and among mutation subtypes grouped according to preference for downstream pathways., Results: a total of 1117 patients were included; 38% had KRAS mutated tumours, 17% had G12C. Among KRAS mutated, G12C was the most frequent mutation in former/current smokers (45%) and G12D in never smokers (46%). There were no significant differences in survival according to KRAS status, G12C status, among KRAS mutation subtypes or mutation preference for downstream pathways., Conclusion: KRAS status or KRAS mutation subtype did not have any significant influence on PFS or OS.

Objective: assessing the prognostic role of miR-20a-5p, in terms of clinical outcome, in a large multi-institutional cohort study., Methods: Tissue microarrays from 535 patients' prostatectomy specimens were constructed. In situ hybridization was performed to assess the expression level of miR-20a-5p in different tissue subregions: tumor stroma (TS) and tumor epithelium (TE). In vitro analysis was performed on prostate cancer cell lines., Results: A high miR-20a-5p expression was found negatively in association with biochemical failure in TE, TS and TE + TS ( p = 0.001, p = 0.003 and p = 0.001, respectively). Multivariable analysis confirmed that high miR-20a-5p expression in TE independently predicts dismal prognosis for biochemical failure (HR = 1.56, 95% CI: 1.10-2.21, p = 0.014). Both DU145 and PC3 cells exhibited increased migration ability after transient overexpression of miR-20a-5p, as well as significant elevation of invasion in DU145 cells., Conclusion: A high miR-20a-5p expression in tumor epithelium is an independent negative predictor for biochemical prostate cancer recurrence.

Background: We previously proposed an immune cell score (tumour node metastasis (TNM)-Immune cell score) classifier as an add-on to the existing TNM staging system for non-small cell lung cancer (NSCLC). Herein, we examined how to reliably assess a tertiary lymphoid structure (TLS) score to refine the TNM staging system., Methods: Using immunohistochemistry (CD8/cytokeratin), we quantified TLS in resected NSCLC whole-tumour tissue sections with three different scoring models on two independent collections (total of 553 patients). In a pilot setting, NanoString gene expression signatures were analysed for associations with TLS., Results: The number of TLSs significantly decreased in stage III patients as compared to stage II. The TLS score was an independent positive prognostic factor, regardless of the type of (semi)-quantification strategy used (four-scale semi-quantitative; absolute count of total TLS; subpopulation of mature TLS) or the endpoint (disease-specific survival; overall survival; time to recurrence). Subgroup analyses revealed a significant prognostic impact of TLS score within each pathological stage, patient cohort and main histological subtype. Targeted gene expression analysis showed that high TLS levels were associated with the expression of B cell and adaptive immunity genes/metagenes including tumour inflammation signature., Conclusions: The TLS score increases the prognostic power in each pathological stage and hence has the potential to refine TNM staging in resected NSCLC.

The TNM classification is well established as a state-of-the-art prognostic and treatment-decision-making tool for non-small cell lung cancer (NSCLC) patients. However, incorporation of biological data may hone the TNM system. This article focuses on choosing and incorporating subsets of tissue-infiltrating lymphocyte (TIL), detected by specific immunohistochemistry and automatically quantified by open source software, into a TNM-Immune cell score (TNM-I) for NSCLC. We use common markers (CD3, CD4, CD8, CD20 and CD45RO) of TILs to identify TIL subsets in tissue micro-arrays comprising tumor tissue from 553 patients resected for primary NSCLC. The number of TILs is automatically quantified using open source software (QuPath). Their prognostic efficacy, alone and within a TNM-I model, is evaluated in all patients and histological subgroups. Compared with previous manual semi-quantitative scoring of TILs in the same cohort, the present digital quantification proved superior. As a proof-of-concept, we construct a TNM-I, using TNM categories and the CD8+ TIL density. The TNM-I is an independent prognosticator of favorable diagnosis in both the overall cohort and in the main histological subgroups. In conclusion, CD8+ TIL density is the most promising candidate marker for a TNM-I in NSCLC. The prognostic efficacy of the CD8+ TIL density is strongest in lung squamous cell carcinomas, whereas both CD8+ TILs and CD20+ TILs, or a combination of these, may be candidates for a TNM-I in lung adenocarcinoma. Furthermore, based on the presented results, digital quantification is the preferred method for scoring TILs in the future., (© The Author(s) 2020. Published by Oxford University Press.)

Introduction: Being diagnosed with cancer and undergoing treatment is a life-changing experience, and many cancer patients find the physical, emotional and social effects of the disease to be stressful. This study explores the experiences of cancer patients and their relatives from all parts of Northern Norway visiting the centralised cancer support centre., Methods: In a comprehensive prospective survey, 286 visitors were invited to participate and 181 of these accepted. The characteristics of the participants, their expectations for visiting the centre, whether they wanted to meet peers or volunteers rather than clinicians and how they viewed the centre in the context of cancer care were evaluated., Results: Most satisfied were visitors aged less than 50 years, women and those reporting a 'strong social network'. The majority of the visitors wanted to have better access to peers (with a similar cancer diagnosis) (89%), cancer nurses (75%) or oncologists (71%). About a third of the participants (29.8%) lived in communities with fewer than 5000 inhabitants and 59.4% in municipalities with fewer than 15 inhabitants/km2. There were no significant differences in the characteristics of the participants, or in their evaluation of the support centre, when stratified by number of inhabitants or population density in their home community., Conclusion: The cancer support centre was highly valued by patients and their relatives for meeting peers. The centre was most frequently visited by and most popular among women and those self-reporting strong social networks. Access to oncology doctors and nurses in this setting could be valuable. Participants living in remote areas had similar characteristics and evaluated the support centre similarly to those living in more urban areas.

Preclinical evidence suggests that stromal expression of platelet-derived growth factor receptors (PDGFRs) stimulates tumor development and diminishes intratumoral drug uptake. In non-small cell lung cancer (NSCLC), the clinical relevance of stromal PDGFR expression remains uncertain. Tumor specimens from 553 patients with primary operable stage I-IIIB NSCLC was obtained and tissue micro-arrays (TMA) were constructed (Norwegian cohort). Immunohistochemistry (IHC) was used to evaluate the expression of PDGFRα and -β in stromal cells and to explore their impact on patient survival. Results were validated in a non-related cohort consisting of TMAs of 367 stage I (A and B) NSCLC patients (Swedish cohort). High stromal PDGFRα expression was an independent predictor of increased survival in the overall populations and SCC (squamous cell carcinoma) subgroups of both investigated cohorts. PDGFRβ was an independent predictor of poor survival in the overall Norwegian cohort and an independent predictor of increased survival in the ADC (adenocarcinoma) subgroup of the Swedish cohort. Tumors displaying the combination PDGFRα-low/PDGFRβ-high exhibited inferior survival according to increasing stage in the Norwegian cohort. This study confirms that high stromal expression of PDGFRα is a predictor of increased survival in NSCLC. Further exploration of the prognostic impact of PDGFRβ and the relationship between PDGFRα and -β is warranted.

Macrophages are important inflammatory cells that regulate innate and adaptive immunity in cancer. Tumor-associated macrophages (TAMs) are thought to differentiate into two main phenotypes: proinflammatory M1 and protumorigenic M2. Currently, the prognostic impact of TAMs and their M1 and M2 phenotypes is unclear in non-small cell cancer (NSCLC). The present study was set up to evaluate an approach for identifying common M1 and M2 macrophage markers and explore their clinical significance in NSCLC. Using multiplex chromogenic immunohistochemistry, tissue microarrays of 553 primary tumors and 143 paired metastatic lymph nodes of NSCLC specimens were stained to detect various putative macrophage phenotypes: M1 (HLA-DR/CD68), M2 (CD163/CD68), M2 (CD204/CD68), and pan-macrophage (CD68/CK). Correlation analyses were performed to examine the relationship between TAMs and adaptive/innate immune infiltrates. HLA-DR + /CD68 + M1 TAM level significantly decreased from pathological stage I to III. In a compartment-specific correlation analysis, moderate to strong correlations were observed between both TAM subsets (M1 and M2) with CD3-, CD8-, CD4-, and CD45RO-positive immune cells. Survival analyses, in both stromal and intratumoral compartments, revealed that high levels of HLA-DR + /CD68 + M1 (stroma, hazard ratio [HR] = 0.73, P = .03; intratumor, HR = 0.7, P = .04), CD204 + M2 (stroma, HR = 0.7, P = .02; intratumor, HR = 0.6, P = .004), and CD68 (stroma, HR = 0.69, P = .02; intratumor, HR = 0.73, P = .04) infiltration were independently associated with improved NSCLC-specific survival. In lymph nodes, the intratumoral level of HLA-DR + /CD68 + M1 was an independent positive prognostic indicator (Cox model, HR = 0.38, P = .001). In conclusion, high levels of M1, CD204 + M2, and CD68 macrophages are independent prognosticators of prolonged survival in NSCLC., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The presence of tumor-infiltrating lymphocytes (TILs) positively impacts the outcome of non-small cell lung cancer (NSCLC) patients. Most previous studies have assessed TILs using different immunohistochemical assays. The purpose of this study was to develop and validate a histopathological scoring model for the assessment of TILs in whole-tissue hematoxylin and eosin (H&E)-stained section slides of NSCLC patients and to evaluate the model in an immunoscore setting. Therefore, TIL was evaluated manually on H&E slides from 537 surgical specimens of primary resected stage I-III NSCLC patients. Using stromal TIL score as a stepwise discrete variable, increasing survival was seen with rising TIL level: disease-specific survival (DSS; P = .008), overall survival (P = .036) and disease-free survival (P = .006). Subgroup analysis revealed that high stromal TILs level was associated with superior DSS (P = .047) in patients with squamous cell carcinoma, but not in patients with adenocarcinoma. Multivariable analysis confirmed that high TIL levels independently predict improved prognosis for all endpoints in the overall cohort. In conclusion, high stromal TIL level is an independent favorable prognostic factor in stage I-III NSCLC patients. The comprehensive histological evaluation conducted in this study may be helpful in streamlining TIL quantification for routine clinical use in a future NSCLC immunoscore setting., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The role of steroid hormones in carcinogenesis of the prostate is to some extent unraveled thorough the effect of androgen deprivation therapy on prostate cancer (PCa) progression. Other members of the steroid hormone family, such as progesterone, are also implicated in PCa, but progesterone's role remains undefined. This study aimed to examine the distribution of progesterone receptor isoforms (PGRA, PGRB) in PCa tissue and their association with clinical endpoints. This was conducted retrospectively by collecting radical prostatectomy specimens from 535 patients. Tissue was analyzed using tissue microarray, where representative tumor areas were carefully selected. Protein expression was evaluated through immunohistochemistry, in stromal and epithelial tissue. Associations between receptor expression and clinical data were considered using statistical survival analyses. Herein, we discovered a solely stromal PGRA- and a stromal and epithelial PGRB expression. Further, a high PGRB expression in tumor tissue was associated with an unfavorable prognosis in both univariate and multivariate analyses: Biochemical failure (HR: 2.0, 95% CI: 1.45-2.76, p < 0.001) and clinical failure (HR: 2.5, 95% CI: 1.29-4.85, p = 0.006). These findings are in agreement with our previous investigation on pan-PGR, indicating that the observed negative effect of PGR is represented by PGRB.

Micro RNAs (miRNA) are small non-coding RNAs that post-transcriptionally regulate gene expression. Dysregulation of miRNA cluster 143/145 has been reported in several malignancies, but their role in non-small cell lung cancer (NSCLC) remains elusive. This study investigates the prognostic impact of miR-143 and miR-145 in primary tumors and metastatic lymph nodes in NSCLC tissue. Tissue from 553 primary tumors and 143 matched metastatic lymph nodes were collected and tissue microarrays were constructed. In situ hybridization was used to evaluate miR-143 and miR-145 expression in tumor epithelial cells and stromal cells in the primary tumors and lymph nodes. In vivo data was supplemented with functional studies of cell lines in vitro to evaluate the role of miR-143 and miR-145 in NSCLC tumorigenesis. In our cohort, stromal miR-143 (S-miR-143) and miR-145 (S-miR-145) expression in primary tumor tissue were independent prognosticators of improved disease-specific survival (DSS) in female (S-miR-143, HR: 0.53, p = 0.019) and male patients (S-miR-145, HR: 0.58, p = 0.021), respectively. Interesting correlations between the miR cluster 143/145 and previously investigated steroid hormone receptors from the same cohort were identified, substantiating their gender dependent significance.

Background: Lymphocyte activation gene-3 (LAG-3) is an immune checkpoint receptor and a putative therapeutic target in non-small-cell lung cancer (NSCLC). We explored the prognostic effect of LAG-3 + tumor-infiltrating lymphocytes (TILs) in primary tumors and metastatic lymph nodes in NSCLC and its potential for inclusion in an immunoscore, supplementing the TNM classification., Materials and Methods: Primary tumor tissue from 553 stage I-IIIB NSCLC patients and 143 corresponding metastatic lymph nodes were collected. The expression of LAG-3 was evaluated by immunohistochemistry on tissue microarrays., Results: On univariate analysis, LAG-3 + TILs in the intraepithelial and stromal compartments of primary tumors and in the intraepithelial and extraepithelial compartments of metastatic lymph nodes were associated with improved disease-specific survival (DSS). On multivariate analysis, stromal LAG-3 + TILs were a significant independent predictor of improved DSS (hazard ratio [HR], 0.59; 95% confidence interval [CI], 0.43-0.82; P = .002). Stromal LAG-3 + TILs did not have prognostic impact across all pathologic stages. In the metastatic lymph nodes, intraepithelial (HR, 0.61; 95% CI, 0.38-0.99; P = .049) and extraepithelial (HR, 0.54; 95% CI, 0.29-0.70; P < .001) LAG-3 + TILs were independently associated with favorable DSS., Conclusion: LAG-3 + TILs are an independent positive prognostic factor in stage I-IIIB NSCLC. LAG-3 in metastatic lymph nodes is a candidate marker for an immunoscore in NSCLC., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Solid tumours need a blood supply, and a large body of evidence has previously suggested that they can grow only if they induce the development of new blood vessels, a process known as tumour angiogenesis. On the basis of this hypothesis, it was proposed that anti-angiogenic drugs should be able to suppress the growth of all solid tumours. However, clinical experience with anti-angiogenic agents has shown that this is not always the case. Reports of tumours growing without the formation of new vessels can be found in the literature dating back to the 1800s, yet no formal recognition, description and demonstration of their special biological status was made until recently. In 1996, we formally recognized and described non-angiogenic tumours in lungs where the only blood vessels present were those originating from normal lung tissue. This is far from an isolated scenario, as non-angiogenic tumour growth has now been observed in tumours of many different organs in both humans and preclinical animal models. In this Opinion article, we summarize how these tumours were discovered and discuss what we know so far about their biology and the potential implications of this knowledge for cancer treatment.

Lung cancer is the leading cause of cancer deaths. Novel predictive biomarkers are needed to improve treatment selection and more accurate prognostication. PAX6 is a transcription factor with a proposed tumour suppressor function. Immunohistochemical staining was performed on tissue microarrays from 335 non-small cell lung cancer (NSCLC) patients for PAX6. Multivariate analyses of clinico-pathological variables and disease-specific survival (DSS) was carried out, and phenotypic changes of two NSCLC cell lines with knockdown of PAX6 were characterized. While PAX6 expression was only associated with a trend of better disease-specific survival (DSS) (p = 0.10), the pN+ subgroup (N = 103) showed significant correlation between high PAX6 expression and longer DSS (p = 0.022). Median survival for pN + patients with high PAX6 expression was 127.4 months, versus 22.9 months for patients with low PAX6 expression. In NCI-H661 cells, knockdown of PAX6 strongly activated serum-stimulated migration. In NCI-H460 cells, PAX6 knockdown activated anchorage-independent growth. We did not observe any significant effect of PAX6 on proliferation in either of cell lines. Our findings strongly support the proposition of PAX6 as a valid and positive prognostic marker in NSCLC in node-positive patients. There is a need for further studies, which should provide mechanistical explanation for the role of PAX6 in NSCLC.

Due to insufficient prognostic tools, failure to predict aggressive prostate cancer (PC) has left patient selection for radical treatment an unsolved challenge. This has resulted in overtreatment with radical therapy. Better prognostic tools are urgently warranted. MicroRNAs (miRs) have emerged as important regulators of cellular pathways, resulting in altered gene expressions. miR-205 has previously been observed downregulated in PC, acting as tumor suppressor. Herein, the expression of miR-205 in prostate tissue was examined in a large, well-described cohort of 535 Norwegian prostatectomy patients. Using in situ hybridization, miR-205 expression was semiquantatively measured in normal and tumor tissues from radical prostatectomy specimens. Associations with clinicopathological data and PC relapse were calculated. Expression of miR-205 was lower in tumor epithelium compared to normal epithelium. No association was observed between miR-205 expression in primary tumor epithelium and cancer relapse. In contrast, high expression of miR-205 in normal epithelium was independently associated with biochemical relapse (HR = 1.64, p = 0.003). A prognostic importance of miR-205 expression was only found in the normal epithelium, raising the hypothesis of epithelial crosstalk between normal and tumor epithelium in PC. This finding supports the proposed novel hypothesis of an anti-cancerogenous function of normal epithelium in tumor tissue.

The immune checkpoint receptor CTLA-4 plays a crucial part in negatively regulating T cell activation and maintaining self-tolerance. It is frequently overexpressed in a variety of malignancies, yet its prognostic impact in non-small cell lung cancer (NSCLC) remains unclear. We constructed tissue microarrays from tumor tissue samples and evaluated the immunohistochemical expression of CTLA-4 in 536 patients with primary resected stage I-IIIA NSCLC. Expression of CTLA-4 was analyzed in tumor and stromal primary tumor tissue and in locoregional metastatic lymph nodes. CTLA-4 expression in neither tumor epithelial cells (T-CTLA-4) nor stromal cells (S-CTLA-4) of primary tumors was significantly associated with disease-specific survival (DSS) in all patients. However, high S-CTLA-4 expression independently predicted significantly improved DSS in the squamous cell carcinoma subgroup (HR 0.62, 95% CI 0.41-0.93, P = 0.021). In contrast, there was an independent negative prognostic impact of T-CTLA-4 expression in metastatic lymph nodes (HR 1.65, 95% CI 1.03-2.65, P = 0.039). Our results indicate that the expression of CTLA-4 has diverging prognostic impacts in metastatic NSCLC lymph nodes versus primary tumors. The presented results highlight important differences in the tumor microenvironments of primary and metastatic NSCLC tissues, and have potential to guide treatment and clinical sampling strategies.

Introduction: To compare the efficacy of silver in situ hybridization (SISH) and immunohistochemistry (IHC) in detecting MET and IGF1R alterations and to investigate their prevalence and prognostic significance. A possible correlation between MET receptor expression, MET gene alterations and the two most frequent occurring EGFR gene mutations was also investigated., Materials and Methods: Stage I to IIIA tumors from 326 patients with NSCLC were immunohistochemically tested for protein expression of MET and IGF-1. Their cytoplasmic expression was compared with the gene copy number of the MET and IGF1Rgenes by SISH in paraffin-embedded, formalin-fixed material. Correlations were made with the immunohistochemical expression of two frequent EGFR mutations and clinicopathological variables. Univariate and multivariate survival analyses was used to evaluate the prognostic efficacy of the tested markers., Results: In univariate analyses, high cytoplasmic MET expression showed a significant negative prognostic effect in adenocarcinoma patients (p = 0.026). MET gene to chromosome 7 ratio was a significant positive prognostic marker (p = 0.005), probably only due to the highly negative prognostic significance of chromosome 7 polysomy (p = 0.002). High IGF1R gene copy number was a negative prognostic marker for all NSCLC patients (p = 0.037). In the multivariate analysis, polysomy of chromosome 7 in tumor cells correlated significantly and independently with a poor prognosis (p = 0.011). In patients with adenocarcinoma, a high cytoplasmic MET expression was an independent negative prognostic marker (p = 0.013). In males a high IGF1R gene copy number to chromosome 15 count ratio was significantly and independently correlated to a poor prognosis (p = 0.018)., Conclusion: MET protein expression provides superior prognostic information compared with SISH. Polysomy of chromosome 7 is an independent negative prognostic factor in NSCLC patients. This finding has an important implication while examining genes located on chromosome 7 by means of SISH. High IGF1R gene copy number to chromosome 15 count ratio is an independent predictor of inferior survival in male patients with primary NSCLC.

Programmed cell death protein 1 (PD-1) and its ligand Programmed death ligand 1 (PD-L1) have gained massive attention in cancer research due to recent availability and their targeted antitumor effects. Their role in prostate cancer is still undetermined. We constructed tissue microarrays from prostatectomy specimens from 535 prostate cancer patients. Following validation of antibodies, immunohistochemistry was used to evaluate the expression of PD-1 in lymphocytes and PD-L1 in epithelial and stromal cells of primary tumors. PD-L1 expression was commonly seen in tumor epithelial cells (92% of cases). Univariate survival analysis revealed a positive association between a high density of PD-1+ lymphocytes and worse clinical failure-free survival, limited to a trend (p = 0.084). In subgroups known to indicate unfavorable prostate cancer prognosis (Gleason grade 9, age < 65, preoperative PSA > 10, pT3) patients with high density of PD-1+ lymphocytes had a significantly higher risk of clinical failure (p = < 0.001, p = 0.025, p = 0.039 and p = 0.011, respectively). In the multivariate analysis, high density of PD-1+ lymphocytes was a significant negative independent prognostic factor for clinical failure-free survival (HR = 2.48, CI 95% 1.12-5.48, p = 0.025).

Introduction: Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) or its ligand, PD-L1, have gained momentum in the treatment of non-small cell lung cancer (NSCLC). However, their prognostic significance remains controversial. The present study evaluated the expression of PD-L1 and PD-1 and their potential role in an Immunoscore, supplementing the TNM classification of NSCLC., Materials and Methods: Tissue microarrays constructed from tumor tissue samples from 2 cohorts of a total of 536 patients (University Hospital of North Norway, n = 285; Nordland Hospital, n = 251) with primary resected stage I to IIIA NSCLC. PD-L1 and PD-1 were evaluated by immunohistochemistry in the primary tumor and metastatic lymph node tissue., Results: In univariate analysis, a high density of PD-L1 + immune cells in the stromal compartment (S-PD-L1) and PD-1 + intraepithelial tumor infiltrating lymphocytes (T-PD-1) was associated with favorable disease-specific survival (DSS; S-PD-L1, P = .004; T-PD-1, P = .012), both limited to the squamous cell carcinoma histologic subgroup (S-PD-L1, P = .002; T-PD-1, P = .034). A combined low S-PD-L1 and T-PD-1 was associated with poor survival in all patients (DSS: hazard ratio [HR], 1.81; 95% confidence interval [CI], 1.37-2.40; P < .001) at both centers and for all pathologic stages. In multivariate analysis, S-PD-L1 and T-PD-1 were independent positive prognostic factors, and combined low scores remained an independent prognosticator for poor survival (DSS: HR, 1.72; 95% CI, 1.29-2.28; P < .001; disease-free survival, P = .001; overall survival, P = .005)., Conclusion: Our study identified S-PD-L1 and T-PD-1 as independent positive prognostic factors for NSCLC patients. Their combination added significant prognostic impact within each pathologic stage and hence are feasible to include in a TNM Immunoscore., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Due to a lack of sufficient diagnostic tools to predict aggressive disease, there is a significant overtreatment of patients with prostate cancer. Platelet derived growth factors (PDGFs) and their receptors (PDGFRs) are key regulators of mesenchymal cells in the tumor microenvironment, and has been associated with unfavorable outcome in several other cancers. Herein, we aimed to investigate the prognostic impact of PDGFR-β and its ligands (PDGF-B and PDGF-D) in a multicenter prostatectomy cohort of 535 Norwegian patients. Using tissue microarrays and immunohistochemistry, the expression of ligands PDGF-B and PDGF-D and their corresponding receptor, PDGFR-β, was assessed in neoplastic tissue and tumor-associated stroma. PDGFR-β was expressed in benign and tumor associated stroma, but not in epithelium. High stromal expression of PDGFR-β was independently associated with clinical relapse (HR = 2.17, p = 0.010) and biochemical failure (HR = 1.58, p = 0.002). This large study highlights the prognostic importance of PDGFR-β expression, implicating its involvement in prostate cancer progression even in early stage disease. Hence, analyses of PDGFR-β may help distinguish which patients will benefit from radical treatment, and since PDGFR-β is associated with relapse and shorter survival, it mandates a focus as a therapeutic target.

There is a need for better prognostication in prostate cancer (PC). "The micromanager of hypoxia", microRNA-210 (miR-210) is directly linked to hypoxia, is overexpressed in PC and has been implied in tumor cell-fibroblast crosstalk. We investigated the prognostic impact of miR-210 in tumor cells and fibroblasts in PC. Tumor and stromal samples from a multicenter PC cohort of 535 prostatectomy patients were inserted into tissue microarrays. To investigate the expression of miR-210, we used in situ hybridization and two pathologists semiquantitatively scored its expression. Overexpression of miR-210 in tumor cells was not associated to biochemical failure-free survival (BFFS, p = 0.85) or clinical failure-free survival (CFFS, p = 0.09). However, overexpression of miR-210 in fibroblasts was significantly associated to a poor CFFS (p = 0.001), but not BFFS (p = 0.232). This feature was validated in both cohorts. Overexpression of miR-210 was independently associated with a reduced CFFS (HR = 2.76, CI 95% 1.25-6.09, p = 0.012). Overexpression of miR-210 in fibroblasts is independently associated with a poor CFFS. This highlights the importance of fibroblasts and cellular compartment crosstalk in PC. miR-210 is a candidate prognostic marker and potential therapeutic target in PC.

Recent data indicate that tumor-associated neutrophils (TANs) serve a dual role in tumor progression and regression. CD66b is a neutrophil marker and has been associated with patient outcome in various cancers. However, its clinical impact in non-small cell lung cancer (NSCLC) remains controversial. 536 NSCLC patients, of which 172 harbored lymph node metastases, were included in this study. Tissue microarrays were constructed and multiplexed immunohistochemistry of CD66b, CD34 and pan-keratin was performed to evaluate the localization and quantity of CD66b+ TANs. High intratumoral CD66b+ TANs density in squamous cell carcinoma (SCC) subgroup was an independent positive prognosticator for disease-specific survival (P = 0.038). In contrast, high intratumoral TANs density was an independent negative prognostic factor in the adenocarcinoma (ADC) subgroup (P= 0.032). Likewise, in ADC patients with lymph node metastases, high level of intratumoral TANs was associated with poor prognosis (P = 0.003). Stromal CD66b+ TANs were not associated with outcome of NSCLC patients. In conclusion, CD66b+ TANs show diverging prognostic effect in NSCLC patients according to histological subgroups. The presence of CD66b+ TANs could prove pivotal for development of an immunoscore in ADC NSCLC patients.

Androgens are considered important in normal prostate physiology and prostate cancer (PCa) pathogenesis. However, androgen-targeted treatment preventing PCa recurrence is still lacking. This indicates additional mediators contributing to cancer development. We sought to determine the prognostic significance of estrogen receptors, ERα and -β, and the aromatase enzyme in PCa. Tissue microarrays were created from 535 PCa patients treated with radical prostatectomy. Expression of ERα, ERβ and aromatase were evaluated using immunohistochemistry. Representative tumor epithelial (TE) and tumor stromal (TS) areas were investigated separately. Survival analyses were used to evaluate the markers correlation to PCa outcome. In univariate analyses, ERα in TS was associated with delayed time to clinical failure (CF) (p = 0.042) and PCa death (p = 0.019), while ERβ was associated with reduced time to biochemical failure (BF) (p = 0.002). Aromatase in TS and TE was associated with increased time to BF and CF respectively (p = 0.016, p = 0.046). Multivariate analyses supported these observations, indicating an independent prognostic impact of all markers. When stratifying the analysis according to different surgical centers the results were unchanged. In conclusion, significant prognostic roles of ERα, ERβ and aromatase were discovered in the in PCa specimens of our large multicenter cohort.

Sex steroids and their receptors are important in the fetal development of normal lung tissue. In addition emerging evidence reveals their significance in lung cancer pathogenesis. This encourages the exploitation of hormone receptors as treatment targets in lung cancer, as it has been successfully used in breast cancer. This study investigates the prognostic impact of estrogen receptor (ER) α and β and the aromatase (AR) enzyme in non-small cell lung cancer (NSCLC) patients. Tumor tissue from 335 NSCLC patients was collected and tissue microarrays (TMAs) were constructed. Immunohistochemical analyses were performed to evaluate the expression of ERα, ERβ and AR in the cytoplasme and nuclei of cells in the tumor epithelial and stromal compartment. By use of survival statistics we investigated the markers impact on disease-specific survival (DSS). Nuclear ERβ expression in tumor epithelial cells in female patients (HR 3.03; 95% CI 1.39-6.61) and tumor cell AR expression in all patients (HR 1.55; 95% CI 1.08-2.23) were significant negative prognostic markers of disease-specific survival in our cohort. High ERβ expression correlates with worse outcome in female patients. Further, patients with high AR expression had an unfavorable prognostic outcome compared with patients expressing low AR levels. These results emphasize the importance of sex steroids role in NSCLC, and, as anti-hormonal drugs are widely available, could lead to the development of novel palliative or even adjuvant treatment strategies in this patient population., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Operable non-small cell lung cancer (NSCLC) patients whose tumours have spread to regional or central lymph nodes at the time of diagnosis have dismal prognoses compared with those who have limited disease. The current TNM staging system for NSCLC poorly distinguishes patients with lymph-node metastases who will succumb to, and those who will eventually be cured from, their disease. This novel study: (1) evaluates the presence of different subsets of intraepithelial tumour-infiltrating lymphocytes (TILs) in lymph nodes with metastases from NSCLC patients; (2) explores the impact of intraepithelial TILs in lymph nodes on survival; (3) correlates their presence with both intraepithelial and stromal TILs in their corresponding primary tumours., Methods: Metastatic lymph-node tissue from 143N+ NSCLC patients was collected and tissue microarrays were constructed. Immunohistochemistry was used to evaluate the presence of intraepithelial CD3+, CD4+, CD8+, CD20+ and CD45RO+ TILs and their impact on survival., Results: A high level of intraepithelial CD45RO+ TILs in lymph-node metastases from N+ NSCLC patients was an independent positive prognostic factor for disease-specific survival in all patients (HR=0.58, P=0.029) and in squamous cell carcinoma (HR=0.31, P=0.006), but not in adenocarcinoma patients., Conclusions: The presence of intraepithelial CD45RO+ cells in lymph-node metastases from N+ NSCLC patients predicts favourable disease-specific survival and outperforms the established TNM staging system in the SCC subgroup.